Distinct effect of calorie restriction between congenic mating types of Cryptococcus neoformans

Cryptococcus neoformans (Cn) is an opportunistic yeast that causes meningoencephalitis in immunocompromised individuals. Calorie restriction (CR) prolongs Cn replicative lifespan (RLS) and mimics low-glucose environments in which Cn resides during infection. The effects of CR-mediated stress can differ among strains and have only been studied in MATα cells. Cn replicates sexually, generating two mating types, MATα and MATa. MATα strains are more dominant in clinical and environmental isolates. We sought to compare the effects of CR stress and longevity regulation between congenic MATα and MATa. Although MATα and MATa cells extended their RLS in response to CR, they engaged different pathways. The sirtuins were upregulated in MATα cells under CR, but not in MATa cells. RLS extension was SIR2-dependent in KN99α, but not in KN99a. The TOR nutrient-sensing pathway was downregulated in MATa strains under CR, while MATα strains demonstrated no difference. Lower oxidative stress and higher ATP production were observed in KN99α cells, possibly due to higher SOD expression. SIR2 was important for mitochondrial morphology and function in both mating types. Increased ATP production during CR powered the upregulated ABC transporters, increasing efflux in MATα cells. This led to enhanced fluconazole tolerance, while MATa cells remained sensitive to fluconazole. Our investigation highlights differences in the response of the mating types to CR.

In the human host, Cn experiences low glucose environments, especially in lung and brain tissues.Given the notable disparity between the prevalence of MATa and MATα cells during human infections, we investigated the impact of CR on the RLS of both mating types in two strains: KN99 (serotype A) and JEC (serotype D) in both standard synthetic media (SM) and calorie-restricted (CR) conditions.
Under standard conditions, the RLS for KN99α and KN99a was comparable (16.5 vs. 13.1 generations) (Fig. 1a).However, CR had a more pronounced pro-longevity effect on KN99a, boosting its mean RLS by a striking 451% (13.1 to 72.3 generations), whereas the RLS increased by 109% in KN99α cells grown under CR (16.5 to 34.6 generations).Similarly, JEC21 (MATα) and JEC20 (MATa) had comparable RLS (11.7 vs 9.3 generations) (Fig. 1b).When subjected to CR, the JEC21 mean RLS saw a 300% increase (11.7 to 47.2 generations), whereas JEC20 saw a RLS extension of 270% with CR (9.3 to 34.4 generations).In KN99α, KN99a, and JEC21, CR also significantly increased the variability of the RLS (stochasticity) when compared to the same strain grown under SM media.These findings demonstrate CR had a significant pro-longevity effect on both mating types in two different Cn strains.

Effect of Sir2 on lifespan extension in MATα and MATa
The highly conserved family of NAD+-dependent histone deacetylase, known as sirtuins, modulate RLS in several organisms [23][24][25] .Recognizing the already established role of SIR2 in CR-mediated longevity of the H99 strain 27 , we compared the transcriptional regulation of the sirtuin family under calorie-restricted growth conditions in both mating types.Notably, SIR2 was similarly upregulated fourfold in KN99α (Fig. 2a) and JEC21 (Fig. 2c) when subjected to CR.Furthermore, CR led to the upregulation of other sirtuins in MATα strains.In KN99α, mRNA levels of HST2 and HST4 were elevated (3.5-fold and 5.5-fold, respectively), whereas in JEC21, HST4 and HST5 mRNA levels were elevated (2.3-fold and 2.5-fold, respectively).In contrast, sirtuins were not regulated in the MATa strains KN99a and JEC20 during CR (Fig. 2b and d).Collectively, these findings suggest that under CR, sirtuin regulation occurs in MATα strains, but not in MATa strains.
To further explore the impact of SIR2 on RLS extension, we employed Δsir2 mutants of both KN99α and KN99a.A KN99α Δsir2-1 strain from the Madhani knockout collection was used.A second KN99α Δsir2-2 mutant and two KN99a Δsir2 mutants were generated employing a TRACE CRISPR-Cas9 system.Successful deletion was verified by PCR of the complete SIR2 gene.In addition, SIR2 mRNA expression by qPCR verified the lack of SIR2 transcription (Fig. S1).Finally, the whole deletion locus was sequenced to confirm proper integration (Fig. S1 and Supplemental Data: Sequencing).Consistent with previous findings, the removal of the SIR2 gene in KN99α led to a notable reduction in RLS in both mutant strains (KN99α Δsir2-1: 25%, 16.5 to 12.4 generations; KN99α Δsir2-2: 30%, 16.5 to 11.6 generations) when compared to the wild-type strain in SM media (Fig. 2e).When placed under CR, both mutants failed to extend their RLS (KN99α Δsir2-1: 12; KN99α Δsir2-2: 11.3 generations).This observation reinforces that CR-induced extension of RLS is contingent upon the presence of SIR2 in KN99α.Contrastingly, in the KN99a strain, loss of SIR2 had no effect on the mean RLS compared to the wild-type in SM media (KN99a Δsir2-1: 13.1 vs 13.1; KN99a Δsir2-2: 13.1 vs 12.6 generations) (Fig. 2f).CR had a substantial pro-longevity effect on the RLS of both KN99a mutants, (KN99a Δsir2-1: 133%, 13.1 to 30.6; KN99a Δsir2-2: 71%, 12.6 to 21.6 generations) although to a lesser extent than wild-type.These findings reinforce that SIR2 is not essential to the pro-longevity effect of CR in KN99a, suggesting it might play a secondary role in MATa strains.
The role of the remaining sirtuins on Cn lifespan was not known.Analysis of the RLS of Δhst2, Δhst3, Δhst4, and Δhst5 mutants indicated no change in RLS relative to the wild-type in SM media, therefore RLS determination under CR was not further pursued (Fig. S2).In summary, these data suggest that SIR2 plays a divergent role in MATa than in MATα when the fungus is grown under CR whereas the other sirtuins do not appear to affect RLS in either mating type.

TOR and PKA pathway dynamics in MATα and MATa cells
Considering that the downregulation of nutrient-sensing pathways also contributes to CR-induced pro-longevity in Sc, we investigated the regulation of TOR and PKA pathway genes in MATa and MATα cells.Our findings showed that, while TOR pathway genes remained unchanged in KN99α under CR (Fig. 3a), JEC21 exhibited minimal upregulation of certain genes such as TOR1, AVO1, AVO2, and TSC2 (Fig. 3c).Conversely, both MATa strains KN99a and JEC20 displayed mostly downregulation of TOR pathway genes (Fig. 3b and d) except TAP42, a downstream effector of the TOR complex that was upregulated in KN99a (Fig. 3b).The Δsir2 mutants of both mating types exhibited no changes in the regulation of TOR pathway-associated genes (Fig. S3), suggesting that the TOR pathway does not compensate for the deletion of SIR2 to increase the RLS.Transcriptional assessments of PKA pathway genes revealed mostly a lack of expression levels in both MATα and MATa cells, except for the downregulation of GPG1 in both mating types and PKA2 in JEC21 and KN99a (Fig. 3e-h).These results demonstrate that the TOR pathway is downregulated only in MATa strains during CR, possibly leading to the CR-mediated RLS extension in MATa strains.

Impact of calorie restriction on mitochondrial function
In Sc, growth under CR is accompanied by mitochondrial biogenesis and increased respiration, resulting in decreased oxidative damage and a prolonged lifespan 30,31 .As the sirtuins have been associated with mitochondrial function and morphology 32,33 , we compared the mitochondrial function of both mating types exposed to SM and CR growth conditions.Changes in mitochondrial morphology are caused by dynamic reorganization of the mitochondrial network and play a pivotal role in responding to glucose starvation.We therefore investigated the mitochondrial morphology utilizing the mitochondrion-specific fluorescent dye MitoTracker Green FM, and deconvolution fluorescence microscopy.These data show distinct differences in mitochondrial morphologies between KN99α, KN99a cells, and their respective Δsir2 mutants.Specifically, tubular mitochondrial networks were observed in both mating types grown in SM medium, while glucose starvation led to fragmentation, which was more pronounced with centralized clustering of mitochondria in KN99a when compared to KN99α.Interestingly, loss of SIR2 was associated with diffuse mitochondrial morphology in both mating types grown in SM and CR conditions (Fig. 4a).Mitochondrial mass was measured by MitoTracker Green FM dye and decreased by 30-50% under CR exposure, whereby the KN99a strain had a lower mass in SM media (Fig. 4b).
Mitochondrial membrane potential (MMP) was measured by staining the cells with the JC-1 dye.MMP regulates respiration rates and correlates with mROS 34,35 .MMP was comparable between KN99α and KN99a cells, and loss of SIR2 was associated with lower MMP.Under CR, MMP increased for all strains except the Δsir2 mutant of KN99a (Fig. 4c).The respiratory process both generates ATP and inadvertently produces mitochondrial reactive oxygen species (mROS) which can be measured by the MitoSOX Red dye.Our data demonstrated higher mROS levels in KN99a compared to KN99α.Consistent with higher MMP, glucose limitation led to a reduction in mROS for both strains, whereas the lower MMP was associated with decreased mROS formation in the Δsir2 mutants (Fig. 4d).It is noteworthy that only KN99α produced increased levels of ATP when grown Pro-longevity Effect is SIR2-dependent in KN99α.CR led to the upregulation of (a) SIR2 (4.07-fold, p = 0.0023), HST2 (3.4-fold, p = 0.014), and HST4 (5.5-fold, p < 0.0001) in KN99α compared to SM media (MATα), while (b) KN99a showed no statistical difference in expression (MATa).Similarly, CR led to the upregulation of (c) SIR2 (4.2-fold, p = 0.0002), HST4 (2.3-fold, p = 0.0029), and HST5 (2.5-fold, p = 0.0008) in JEC21 compared to SM media (MATα) while (d) JEC20 showed no statistical difference in expression (MATa).qPCR was used to analyze expression and ACT1 was used as an internal control.The dotted lines signify a twofold up-or downregulation of the genes.qPCR was performed in biological triplicate and error bars signify standard deviation between samples.(e) Deletion of SIR2 in KN99α (KN99α Δsir2-1 and KN99α Δsir2-2) shortened RLS compared to the parental strain (p = 0.0024 and p = 0.028).CR-induced pro-longevity effect of KN99α was lost on the mutant strain (KN99α Δsir2 CR; p < 0.0001).(f) Deletion of SIR2 in KN99a (KN99a Δsir2-1 and KN99a Δsir2-2) had no change in RLS compared to the parental strain.CR extended the RLS of the mutant strains (p < 0.0001 and p = 0.0022) but to smaller extents than the parental strain.Statistical analysis was performed with Two-Way ANOVA and Student's t-test with Welch's correction.under CR (145 nM to 284 nM), while no augmentation of ATP levels was seen in KN99a or the Δsir2 mutant strains (Fig. 4e).These data suggest increased oxidative damage under CR stress in KN99a may impair ATP production.Corroborating this interpretation, CR-induced upregulation of both superoxide dismutases (SOD) was found in KN99α (SOD1: 11.3-fold, SOD2: 5.3-fold), whereas CR in KN99a resulted in minimal upregulation of cytoplasmic SOD (SOD1: 2.2-fold) and no differential regulation of mitochondrial SOD.In the KN99α Δsir2 mutant, moderate upregulation of SOD1 (twofold) and SOD2 (2.3-fold) was observed during CR, while no differential regulation was observed in the KN99a Δsir2 mutant (Fig. 4f).Lastly, to gain further insight into the metabolism of the two mating types, we analyzed the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as a measure of glycolysis and oxidative (f) PKA pathway genes GPG1 (0.31-fold, p < 0.0001) and PKA2 (0.48-fold, p = 0.0005) were downregulated, while RAS2 (2.5-fold, p < 0.0001) was upregulated in KN99a during CR compared to SM media.(g) PKA2 (0.37-fold, p = 0.0004) was downregulated in JEC21 in CR growth conditions compared to SM media.(h) GPG1 (0.21-fold, p < 0.0001) was downregulated in JEC20 in CR growth conditions compared to SM media.qPCR was used to analyze expression and ACT1 was used as an internal control.The dotted lines signify a twofold up-or downregulation of the genes.qPCR was performed in biological triplicate and error bars signify standard deviation between samples.Statistical analysis was performed with Two-Way ANOVA.phosphorylation, respectively.KN99a exhibited lower basal metabolism in both ECAR and OCR measurements (Fig. 4g and h), indicating lower rates of glycolysis and respiration.The injection of glucose increased metabolism in both strains, with KN99a CR showing slower glucose metabolization.Inhibition of respiration by Rotenone and Antimycin A (Rot/AA) reduced respiration rates of both strains (Fig. 4h) and revealed that KN99a has lower glycolysis levels compared to KN99α (Fig. 4g).No difference in glycolysis rate was found between SM and CR growth conditions..Given the differences in ATP production between mating types, we compared the FLC tolerance between the two mating types.In contrast to KN99α, and despite comparable baseline susceptibility, the FLC tolerance of the MATa strains KN99a or JEC20 did not change when grown in CR conditions (Fig. 5a).
Since there is a difference in ATP production when SIR2 is deleted, we further analyzed FLC susceptibility in the KN99α Δsir2 mutant.Deletion of SIR2 in KN99α led to a higher susceptibility to FLC under CR, although 80% inhibition was not attained (Fig. S4).

Comprehensive transcriptome analysis in MATα and MATa cells under CR
Lastly, an RNAseq of KN99α and KN99a cells grown under SM and CR was performed to uncover additional differences in CR-induced regulation.Consistent with attenuated growth, CR led to more downregulation of gene transcription in both mating types.KN99α cells altered transcription of 1,580 genes under CR, while KN99a altered transcription of 2415 genes (Fig. 6a).Although both mating types shared 1090 genes, many differentially expressed genes (DEGs) were mating type-specific (Fig. 6b).Heatmap analysis confirmed correlation and clusters in duplicates (Fig. 6c).Gene ontology (GO) classification and enrichment analysis showed different responses to CR.In KN99α, CR led to an increase in 55 molecular functions and 45 biological processes, and a decrease in 27 functions and 52 processes whereas under CR, KN99a increased 46 functions and 39 processes but decreased 65 functions and 90 processes (summarized in Fig. S5).
While overlapping GO categories such as transport, calcium homeostasis, and phosphorylation were noted, the majority of enriched categories were either specific to KN99α or KN99a.For KN99α, upregulated GO categories included NAD/NAD+ binding, which correlates with the activity of NAD+-dependent sirtuins.Furthermore, antioxidant activity, ROS metabolic processes, as well as superoxide dismutases, catalases, and cytochrome c peroxidases that mediate the breakdown of H 2 O 2 were upregulated (Fig. S5).In KN99a, categories related to lipid metabolism, such as fatty acid synthase activity, cellular lipid metabolism, sphingolipid metabolism, and lipid glycosylation, were significantly enriched (Fig. S5).Although the genes of catalase and cytochrome c peroxidase were upregulated, the antioxidant activity GO category was downregulated in KN99a, which included sulfiredoxin, cytochrome c peroxidase, catalase, peroxiredoxin, thioredoxin reductase, and thiol-specific antioxidant protein 3 genes (Fig. S5).Importantly, CR induced altered transcription of many mitochondrial genes in both mating types.In KN99α, 43 mitochondrial genes related to the electron transport chain and ATP synthesis were upregulated.These included 17 genes encoding for subunits of Complex I (NADH dehydrogenase), 4 for Complex II (succinate dehydrogenase), 3 for Complex III (cytochrome bc), one for Complex IV (cytochrome c oxidase) and Complex V (ATP synthase) that were upregulated.Most of the 10 mitochondrial genes downregulated in KN99α by CR were associated with mitochondrial transport.In KN99a, 34 upregulated mitochondrial genes included 8 encoding for genes of Complex I, 3 for Complex II, and 2 for both Complex III and Complex IV subunits.Interestingly, 2 genes encoding Complex I and one for Complex IV subunits were downregulated, along with 5 genes encoding for the ATP synthase of Complex V. CR also increased the transcription of 13 mitochondrial transporter genes, while decreasing the transcription of 8 transporters and 3 genes related to mitochondrial morphology in KN99a.Together, the present findings provide further evidence that MATα and MATa cells respond differently to CR stress.The mating types show distinctly different transcriptomes, including differences in the regulation of mitochondrial function and, specifically, ATP synthesis.

Discussion
The present study addressed the divergent responses of Cn mating types to low glucose stress conditions.We found that Cn MATα cells upregulated SIR2 and other sirtuins, while MATa cells downregulated the TOR pathway, suggesting that the pro-longevity effect in the setting of CR is differently regulated between mating types.This difference in regulation impacts mitochondrial function and drug resistance in a mating type-dependent manner.KN99a cells exhibit higher oxidative damage with increased mROS resulting in compromised mitochondria function and decreased ATP production.As a result, MATa does not exhibit enhanced FLC tolerance in a glucose-limiting environment.These results suggest that MATa cells display inferior stress and pro-longevity response to CR, possibly explaining why this mating type is less prevalent.
While lifespan extension is a common outcome of CR, it is not universal.In fact, both shortened and unchanged RLS have been observed in Cn and other yeast species 37 .In Sc, studies using strains PSY316 and BY4742 have demonstrated RLS extension under CR 28,[38][39][40] , while investigations involving the W303R strain www.nature.com/scientificreports/have yielded different results 41 .Previous studies have also documented variable RLS responses in clinical Cn MATα strains when subjected to CR 37 .Notably, the pro-longevity effect of CR in most of the investigated Cn strains appears more pronounced compared to Sc, with RLS extension reaching up to 400%.This substantial difference may be attributed to differences in glucose metabolism of the two yeasts.Unlike Crabtree-positive Sc, which switches from fermentation to respiration in the presence of oxygen and low glucose concentrations, Crabtree-negative Cn primarily relies on respiration, requiring finer control over their metabolic responses 38,42 .Sir2 and its homologs have an established role in modulating lifespan in Sc and Cn 23,43 .In contrast to Sc, where Sir2 regulates chromatin silencing at the mating locus, telomeres, and rDNA repeats, in Cn, Sir2 predominantly associates with tRNA genes and the rDNA region, with no interaction with the MAT locus or telomeric regions [44][45][46][47] .Our findings highlight differences in Sir2 regulation between mating types of Cn exposed to CR.While transcription of SIR2 and other sirtuins was upregulated in KN99α under CR, it did not affect their transcription in KN99a.Additionally, CR-induced RLS prolongation was SIR2-dependent only in KN99α, and not in KN99a, where Δsir2 continued to exhibit a prolonged RLS under CR.This result confirmed previous findings that H99 RLS extension is SIR2-dependent under low glucose conditions 27 .These findings may indicate that, unlike KN99α, SIR2 activity is not necessary for longevity in KN99a, playing only a secondary role.Similar findings have been described in Sc strains where the pro-longevity effect of CR is only SIR2-dependent in W303R but not in PSY316 23,48 .This data confirms that not all strains are SIR2-dependent for RLS extension, although no correlation with mating types was described in Sc.
The role of other sirtuins was entertained.For Cn Δsir2, Δhst3, and Δhst4 phenotypical changes and hypovirulence have been reported 47 .Unlike SIR2, the deletion of HST3 and HST4 did not affect RLS, indicating that they alone do not play a role in longevity.Since the sirtuins are gene silencers, it has been proposed in Cn that SIR2 deletion leads to loss of heritable epigenetic information 47 , explaining the reported failure to successfully complement their knockout mutants to restore wild-type activity.To account for the lack of complemented strains, we generated two independent Δsir2 mutants for KN99α and KN99a, which demonstrated that the phenotype was consistent.
Decreased TOR activity in Sc, by null mutants or during induction by CR, prolongs their lifespans 28,49 .Consistent with lack of SIR2 regulation we found the TOR pathway was downregulated in MATa strains, but mostly unchanged in MATα strains.This suggests diverse roles of the TOR pathway in the longevity regulation of MATa and MATα strains.Our data supports the concept that SIR2 regulation is not the only response option to CR.While MATα cells are dependent on the function of SIR2 for RLS extension, MATa cells may be dependent on the TOR pathway downregulation.Similar to SIR2, the pro-longevity effect of decreased TOR activity appears independent of mating type in Sc 28,48 .Unfortunately, Cn cannot be studied through a genetic approach because the singular TOR1 gene of Cn is essential, unlike Sc which has two TOR genes 50 .
Mitochondrial dysfunction is a hallmark of aging, as damaged mitochondria are asymmetrically segregated to mother cells, which is associated with higher ROS levels and a lower redox potential 51 .Increased mitochondrial biogenesis and respiration, as well as decreased oxidative stress, are achieved through CR in Sc 30,31 .In contrast to other yeasts, glycolysis and respiration are highly interdependent in Crabtree-negative Cn 52 .As a result of its metabolic inflexibility, Cn needs to fine-tune respiration and cannot live without mitochondrial function 38,42 .Under glucose restriction, KN99a exhibited lower metabolic activity than KN99α, especially when glycolysis was analyzed with Seahorse.Since Cn cannot regenerate NAD+ through fermentation, the cells are unable to meet their ATP demands, resulting in stalled glycolysis 52 .Usually, NAD+ is reduced into NADH during the glycolytic metabolic process, which will generate ATP 53 .Lower glycolysis levels may be the result of a smaller NAD+ pool, which would generate less ATP in KN99a cells.Increased NAD+ binding GO term in KN99α could also indicate higher NAD+ availability.Depletion of cytoplasmic NAD+ can block glycolysis 54 and cause mitochondrial dysfunction, with a decline in energy production and ROS accumulation, producing high oxidative stress 55 .
In the mitochondria of Cn, oxygen is essential for oxidative phosphorylation and ATP production.Furthermore, mitochondrial impairment can lead to oxygen not receiving a full complement of electrons for reduction, ultimately resulting in harmful ROS accumulation 56,57 .Higher MMP levels in Cn under CR indicate sufficient mitochondrial health to facilitate enhanced production of ATP 58 .However, CR-driven increase in ATP production was only observed in KN99α and not in KN99a cells.The RNAseq results explain these findings.During CR, subunits of all electron transport chain complexes were found to be upregulated in KN99α.Despite the upregulation of genes that encode subunits of complexes I-IV, KN99a did not enrich any GO terms related to mitochondrial respiration.This is likely the consequence of the downregulation of genes encoding for subunits of complex V, which is responsible for ATP synthesis in mitochondria.mROS levels were also consistently higher in KN99a compared to KN99α, indicating marked differences in mitochondrial function between mating types.The SODs catalyze the breakdown of ROS into hydrogen peroxide, which is then converted into oxygen and water 59 .Higher mROS levels in KN99a were consistent with lower transcription of mitochondrial and cytoplasmic SOD compared to KN99α.Lower mROS levels in KN99α under CR can be attributed to the upregulation of two SODs, two cytochrome c peroxidases, and a catalase.KN99a transcriptome indicated enhanced transcription of hydrogen peroxide metabolism-associated genes, and downregulation of antioxidant activity, suggesting a compromised antioxidant response.
During permissive, non-stressful growth, the Cn mitochondria are present primarily in a reticular morphology 60,61 .Upon encountering mitochondrial stress, such as CR, mitochondria undergo fusion and fission, enriching tubular and fragmented morphologies 61 .Mitochondrial fusion permits sharing of mitochondrial DNA, and mitochondrial fission facilitates removal of damaged mitochondria.The mitochondria of KN99α underwent fragmentation to counteract the stress of CR, but the mitochondria of KN99a displayed fragmentation with a clustered morphology consistent with more pronounced stress.Taken together, these phenotypes demonstrate that MATa cells display higher oxidative stress and mitochondrial damage than their MATα counterparts.As mitochondria are essential for Cn survival, these differences in stress response may influence mating type prevalence.Deletion of SIR2 influenced mitochondrial function and morphology in both mating types.The Δsir2 mutants displayed an extremely diffuse phenotype, with loss of the tubular and fragmented phenotypes observed in the wild-type strains.This is consistent with the association of mammalian sirtuins with the modulation of fusion/ fission of the mitochondrial network.Inhibition of SIRT1 and SIRT3 alters the mitochondrial morphology by preventing fusion/fission 33,62 .Morphology regulation is achieved by modulation of the fission dynamin-1-like protein (Drp1) and actin cytoskeleton 33,[63][64][65] .SIR2 deletion also led to decreased MMP, ROS, and ATP production.Mammalian sirtuins enhance oxidative phosphorylation, electron transport chain, and ATP formation 32,66,67 , which is consistent with the diminished mitochondrial function observed in Δsir2 mutants of Cn.
Mitochondrial ATP hydrolysis is the energy source for the ABC transporters superfamily.Mitochondrial respiratory activity is directly associated with efflux pump-mediated resistance in several fungi 22,68 .Generally, an increase in respiration leads to a higher ATP generation and transport to the cytoplasm, ensuring that drug efflux pumps gain more available ATP to increase efflux 68 .Compromised ATP production resulted in reduced efflux pump activity in KN99a, explaining why CR did not induce higher FLC tolerance in both MATa strains, in contrast to MATα Cn strains.These results further indicate that MATa strains respond to stress less effectively than MATα, as they can't counteract the effect of FLC.
Congenic MATa/MATα mating pairs are genetically identical except for their mating locus; therefore, this locus could explain the distinct responses to CR between mating types.The MAT locus harbors three transcription factors associated with carbon and nutrient metabolism, ZNF1α/a, STE12α/a, and SXI1α/2a.In Cn, published RNAseq data found STE12α upregulated in H99 (MATα) under CR (fold-change = 10.6) 27, which we confirmed here in KN99α, but not in KN99a.In Cn, Ssn8 is the downstream target of STE12α and, in response to nutritional stress, regulates 75 genes involved in nutrient scavenging 16 .Deletion of STE12α in Cn resulted in decreased activity of SOD enzymes, suggesting a link between the MAT locus and SOD regulation 69 , which could explain the difference in ROS levels between mating types.Differential regulation of these transcription factors or their downstream targets may contribute to several differences between mating types.
In summary, CR exerts a diverse influence on the longevity regulation of Cn mating types, with MATα cells being SIR2-dependent and MATa cells being SIR2-independent, possibly relying on the downregulation of the TOR pathway.This difference in CR response affects mitochondrial function, morphology, and drug resistance in a mating type-dependent manner.MATa strains exhibit higher levels of oxidative stress and mitochondrial damage, as well as a decreased ability to combat antifungal drugs under CR stress.It is possible that better stress response mechanisms could be responsible for the higher prevalence and greater virulence of MATα cells.Our data further encourage efforts to characterize longevity and stress responses in Cn cells.

Deletion of SIR2 in KN99a and KN99α
SIR2 deletion was performed using the TRACE CRISPR-Cas9 system as previously described [70][71][72] .The Cas9 cassette was amplified from the codon-optimized pMH21 plasmid (pRS316-P TEF1 -CAS9_optimized; oligonucleotides M13 F and M13 R) with an Ex Taq DNA-Polymerase.To make the sgRNA construct, the Cryptococcus native U6 promoter was amplified from the BHM2329 plasmid (pRS316-promoterCnU6-sgRNAscaffold-6T; oligonucleotides C8573 and sgRNA R).The reverse oligo contains a 20 bp reverse complement sequence of the genomic target for the Cas9 enzyme (gRNA).The gRNA (upstream of PAM and excludes PAM from the oligonucleotide) was designed using the Eukaryotic Pathogen CRISPR guide RNA/DNA Design Tool (http:// grna.ctegd.uga.edu/) by searching for a sequence within 150 bp of the 5ʹ UTR region up to the start codon of SIR2.The scaffold/terminator sequence was also amplified from the BHM2329 plasmid (oligonucleotides sgRNA F and C8574) with a forward oligo that contains the forward sequence of the gRNA.All fragments were run in a 0.8% agarose gel and gel purified using the QIAquick Gel Extraction Kit.Equal volumes of the U6 and scaffold/ terminator fragments were mixed and used as a template for a fusion PCR using Ex Taq Polymerase (oligonucleotides C8573 and C8574).The resulting fragment was gel extracted and purified.The DNA construct to be integrated was designed to contain 50 bp of homology to the 5ʹ UTR of SIR2 + HYG R (hygromycin resistance gene) + 50 bp of homology to the 3ʹ UTR of SIR2.This was achieved by amplifying the HYG R gene from the pPZP-HYG plasmid with oligonucleotides containing the homology arms to SIR2.The fragment was gel extracted and purified.Transformation by electroporation was performed by growing KN99a and KN99α overnight and inoculating them into 100 mL YPD (OD 600 = 0.2).They were grown for 4-5 h (OD 600 = 0.6-1.0),centrifuged, and washed twice in ice-cold water.The cells were then resuspended in 10 mL of ice-cold electroporation buffer (EB; 10 mM Tris-HCl pH 7.5, 1 mM MgCl 2 , 270 mM sucrose) with 1 mM DTT and incubated on ice for 1 h.Cells were centrifuged and resuspended in 220-250 μL of EB with 1 mM DTT.PCR products were mixed with the cells as follows: 50 μL of cells, 2 μg of Cas9 cassette, 700 ng of sgRNA cassette, and 4 μg of deletion cassette.They were transferred to a pre-cooled 2 mm gap electroporation cuvette, and a negative control with only cells was also transferred into a separate cuvette.Cells were transformed in a MicroPulser Electroporator at the following settings: 2 kv, 4 ms.Following transformation, cells were recovered in 1 mL of YPD media and incubated at 30 °C for 1-2 h.Cells were plated in HYG YPD agar and incubated for 2-3 days.Positive colonies were passaged in selective media three times.For confirmation, DNA was extracted, and PCR was performed to confirm gene deletion (oligonucleotides SIR2 F and SIR R).Additionally, expression was determined by qPCR (oligonucleotides qPCR SIR2 F and qPCR SIR2 R).Oligonucleotides are described in Table S2.

Replicative lifespan analysis
RLS was determined as previously established 73 .Briefly, cells were grown on SM or CR agar plates overnight.Individual 20-30 naïve cells were dissected under a tetrad dissection Axioscope A1 microscope.The RLS is defined as the number of times a mother cell buds before undergoing senescence, which is determined as 24 h without a budding event.

Antifungal susceptibility
The minimum inhibitory concentration (MIC) was determined as previously described 74 .Briefly, 10 5 cells/well of Cn strains were incubated with serially diluted fluconazole (FLC) for 4 days at 37 °C.The MIC was defined as the concentration that inhibits 80% of cell growth (MIC 80 ) at OD 600 .

Transcriptome analysis
Cells were grown overnight in SM or CR media.RNA extraction, cDNA conversion, and qPCR were performed as previously described 36 , following the manufacturers' guidelines.The oligonucleotides employed to analyze the gene expression of the sirtuins, TOR pathway, PKA pathway, SOD, and efflux pumps genes are described in Table S2.The housekeeping gene ACT1 was used as an internal control.Data were calculated by the 2 −ΔΔCT method, as previously described 75 .Expression was considered significant above or below a twofold change threshold.The assay was conducted with 3-5 biological replicates.

RNAseq analysis
KN99α and KN99a were grown overnight in SM or CR media.RNA was extracted using the RNAeasy Plus kit, following the manufacturer's guidelines.RNASeq was performed by Novogene Co. Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads and converted to cDNA.Libraries were clustered and sequenced on an Illumina NovaSeq 6000 platform, which utilizes a paired-end 150 bp sequencing strategy (PE150) according to the manufacturer's instructions.Raw data were processed by removing reads containing adapters, reads containing unknown nucleotides, and low-quality reads.Clean reads were aligned to the reference genome using Hisat2 v2.0.5.Differential expression analysis was performed employing the DESeq2 R package (1.20.0).Genes with a p-value < = 0.05 and |log2FoldChange|> = 0.5 were assigned as differentially expressed.Gene ontology (GO) Enrichment analysis was performed by running differentially expressed genes through FungiDB, selecting for Biological Process (BP) and Molecular Function (MF) with a p-value cutoff of 0.05.Venn diagrams were generated using the jvenn software (http:// jvenn.toulo use.inra.fr/ app/ index.html).Heatmap was generated by Novogene Co.

Cellular ATP levels analysis
Measurement of cellular ATP levels was performed using ATP Bioluminescence Assay Kit following the manufacturer's guidelines.Briefly, cells were cultured overnight in SM and CR media, and 10 7 cells were employed in this assay.Cellular ATP was isolated using an alkaline lysis buffer and 5% trichloroacetic acid.They were vortexed for 15 min in the presence of sterile acid-washed glass beads to initiate lysis.The cell lysates were boiled for 10 min at 100 °C.A standard curve was produced using an ATP Standard, following the manufacturer's guidelines.The samples and standard curve were mixed with a reaction mixture containing Assay Buffer, Substrate, and ATP Enzyme.The luminescence was immediately read on a plate reader.This assay was performed in triplicate.

Measurement of mitochondrial activity and morphology
Mitochondrial reactive oxygen species (mROS) was measured by growing cells overnight in SM and CR media and staining 10 7 cells with MitoSOX Red following the manufacturer's guidelines as previously described, with modifications 22 .Cells were incubated with a final MitoSOX concentration of 5 μM.Mitochondrial membrane potential (MMP) was measured by growing cells overnight in SM and CR media and staining 10 7 cells with JC-1 following the manufacturer's protocol.JC-1 was resuspended in 383 μL of DMSO, at a concentration of 20 mM.The cells were resuspended in a final JC-1 concentration of 200 μM and incubated at 37 °C in the dark for 30 min.After staining, the cells were washed 3× with PBS, and 200 μL were loaded into a black clear-bottom 96-well plate.Fluorescence was measured at λ ex of 535 nm and λ em of 590 nm.Unstained cells were used as a negative control.Mitochondrial mass was measured by growing cells overnight in SM and CR media and staining 10 6 cells with MitoTracker Green FM following the manufacturer's protocol as previously described 22 .All assays were performed in biological triplicate.MitoTracker Green was further used to analyze mitochondrial morphology as previously described 22 .

Rhodamine 6G efflux assay
KN99α and KN99a were grown overnight in SM and SM CR media and Rhodamine 6G assay was performed as previously reported 36 using 2 × 10 7 cells in a biological triplicate.Cells were incubated with 10 μM Rhodamine 6G for 30 min at 37 °C.Efflux was measured after 30 min.

Figure 4 .
Figure 4. CR Affects Mitochondrial Function.(a) Cells stained with MitoTracker Green dye were observed in deconvoluted fluoresce studies.In SM, cells displayed tubular networks.In CR, mitochondria of KN99α (MATα) cells fragmented, and those of KN99a (MATa) cells fragmented and clustered.SIR2 deletion led to dispersed morphologies in both conditions.(b) KN99a exhibited lower mitochondrial mass than KN99α in SM media (p < 0.0001).CR lowered mitochondrial mass in all strains (p < 0.0001) as measured by MitoTracker Green dye.(c) Mitochondrial Membrane Potential (MMP) was comparable between KN99α and KN99a in SM.CR increased MMP in KN99a (p = 0.0001) and KN99α (p < 0.0001), as measured by JC-1 dye.SIR2 deletion decreased MMP in both strains (p < 0.0001 and p = 0.0197).(d) Under CR, mitochondrial reactive oxygen species (mROS) levels were lower in KN99α (p < 0.0001) and KN99a (p = 0.0001) compared to SM, as measured with MitoSOX Red dye.Overall, mROS levels were higher in KN99a cells (SM p = 0.0003, CR p < 0.0001).SIR2 deletion decreased mROS formation (p < 0.0001).(e) ATP levels were increased in KN99α under CR compared to SM (p = 0.0003), while ATP levels were unchanged in all other strains as measured by an ATP Bioluminescence assay Kit.(f) CR upregulated the cytoplasmic superoxide dismutase (SOD1) in KN99α (11.3fold, p = 0.0063), KN99a (2.2-fold, p < 0.0001), and KN99α Δsir2 (2.05-fold, p = 0.0075) compared to SM.The mitochondrial SOD (SOD2) was upregulated only in KN99α (5.39-fold, p = 0.027) and KN99α Δsir2 (2.32-fold, p = 0.0012) during CR.qPCR was used to analyze transcription and ACT1 was the internal control.The dotted lines signify a twofold up-or downregulation of the genes.(g,h) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) profiles were generated by injection of glucose, Rotenone and Antimycin A (Rot/AA), and 2-deoxyglucose (2DG).KN99a has a lower basal metabolism and glycolysis than KN99α.Assays were performed in biological triplicate and error bars signify standard deviation between samples.Statistical analysis was performed with Student's t-test with Welch's correction and One-Way ANOVA.

Figure 6 .
Figure 6.Transcriptomic Changes During Calorie Restriction are Different between KN99α and KN99a.RNAseq of KN99α (MATα) and KN99a (MATa) cells was performed under SM and CR growth conditions.(a) KN99α under CR differentially expressed 1580 genes (759 upregulated and 821 downregulated), while KN99a differentially expressed 2415 genes (1166 upregulated and 1249 downregulated).(b) Venn diagram of differentially expressed genes (DEG) between KN99α and KN99a demonstrated that 1090 DEGs were common between the two strains, while 1323 were unique to KN99a and 488 were unique to KN99α.Image generated with jvenn software.(c) The heatmap showed that duplicates of the same sample (identified by 1 and 2 at the bottom axis) clustered together and that CR samples have a distinctly different expression profile than SM-grown cells. https://doi.org/10.1038/s41598-024-69087-ywww.nature.com/scientificreports/

dependent tolerance to FLC to MATα and MATa under CR
Mitochondria provide the majority of cellular ATP, which fuels the energy-dependent ATP-binding cassette (ABC) transporters.Earlier studies with KN99α have indicated that with advanced generational age or when exposed to CR, Cn exhibits augmented tolerance to FLC mediated by enhanced efflux pump activity of the ABC transporters Vol.:(0123456789) Scientific Reports | (2024) 14:18187 | https://doi.org/10.1038/s41598-024-69087-ywww.nature.com/scientificreports/Effect of age-